A. Borrelia burgdorferi, the agent of Lyme disease, survives and proliferates in both an arthropod vector and various mammalian hosts. During its transmission/infective cycle, B. burgdorferi encounters environmental challenges specific to those hosts. One challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), nitrogen dioxide (NO2), nitrogen trioxide (N2O3) and peroxynitrite (NO3). There are two stages in the infective cycle when B. burgdorferi is exposed to ROS/RNS. The first is during the initial stages of infection of the mammalian host when cells of the immune system attempt to limit and eliminate B. burgdorferi using several mechanisms including the production of ROS and RNS. Surprisingly, the second ROS/RNS challenge occurs during feeding and as the bacteria migrate through the tick salivary during transmission. In FY 2016, we have demonstrated that the salivary glands and midgut of Ixodes scapularis contained significant levels of ROS and RNS during feeding. We compared the ability of B. burgdorferi strains harboring mutations in DNA repair genes that are hypersensitive to killing by ROS or RNS to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks. We showed that the methyl-directed mismatch repair (MMR) gene mutS1 and the nucleotide excision repair (NER) gene uvrB are dispensable for infection of mice, while uvrB promotes the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggested that B. burgdorferi must withstand cytotoxic levels of RNS produced during infection of I. scapularis ticks (1). B. Lyme disease is a vector-borne illness that requires pathogenic strains of B. burgdorferi to adapt to distinctly different environments in its tick vector and various mammalian hosts. Effective colonization (acquisition phase) of a tick requires the bacteria to adapt to tick midgut physiology. Successful transmission (transmission phase) to a mammal requires the bacteria to sense and respond to the midgut environmental cues and up-regulate key virulence factors before transmission to a new host. In FY 2016, we identified one environmental signal that appears to affect both phases of the infective cycle: osmolarity. While constant in the blood, interstitial fluid and tissue of a mammalian host (300mOsm), osmolarity fluctuates in the midgut of feeding Ixodes scapularis. Measured osmolarity of the blood meal isolated from the midgut of a feeding tick fluctuates from an initial osmolarity of 600 mOsm to blood-like osmolarity of 300 mOsm. After feeding, the midgut osmolarity rebounded to 600 mOsm. These changes affect the two independent regulatory networks that promote acquisition (Hk1-Rrp1) and transmission (Rrp2-RpoN-RpoS) of B. burgdorferi. Increased osmolarity affected morphology and motility of wild-type strains, and lysed Hk1 and Rrp1 mutant strains. At low osmolarity, Borrelia cells express increased levels of RpoN-RpoS-dependent virulence factors (OspC, DbpA) required for the mammalian infection. Our results strongly suggest that osmolarity is an important signal that allows the bacteria to adjust gene expression during the acquisition and transmission phases of the infective cycle of B. burgdorferi (2). C. B. burgdorferi's ability to adapt and survive in very different environments (tick versus mammal) is attributed to its ability to sense changes in temperature, pH, cell density, oxygen, manganese and/or exposure to host factors and alter gene expression accordingly. Previous reports have demonstrated that central to the regulation of these responses are the sigma factors, RpoN and RpoS. Importantly, RpoN-dependent regulation of RpoS is responsible for the expression of key virulence factors e.g., outer surface protein C (OspC), OspA and decorin-binding protein A (DbpA) required for infectivity and transmission during the infective cycle. The activities of RpoN are tightly controlled and require ATP-dependent activation. Many of the activators of RpoN-RNA polymerase (RNAP) are response regulators (RR) of two-component regulatory systems and phosphorylation of these RR results in their activation. These RRs are phosphorylated by small molecular weight phosphate donors (e.g., ATP; designated as auto-phosphorylation) or, more commonly, by their cognate protein histidine kinase (HK) in response to an environmental signal. The activator of RpoN in B. burgdorferi, Rrp2 encoded by bb0763 (rrp2), is also a RR of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, Hk2 encoded by bb0764 (hk2). These regulatory components form the Rrp2-RpoN-RpoS signaling cascade that coordinates the expression of virulence factors required for successful transition of B. burgdorferi from its arthropod vector to mammalian hosts. Activation of Rrp2 is essential to initiate this regulatory pathway. The intracellular signal and phosphorylation processes triggering Rrp2 activation are poorly understood. Published reports have suggested that acetyl-phosphate (AcP), a global regulatory molecule in many bacteria, serves as a signal and high energy phosphate donor for Rrp2 activation. In FY2016, we determined that AcP is solely generated from a unique pathway that uses acetate to synthesize undecaprenyl phosphate (C55-P), an essential lipid for cell wall biogenesis. To study this system and the role of acetyl-P as a global regulatory signal, we generated mutants that eliminate acetyl-P synthesis and utilization. Our analyses demonstrate that AcP does not directly modulate Rrp2-RpoN-RpoS regulation nor act as a global regulator in B. burgdorferi (3).